During learning and development, neural circuitry is refined through changes in synapse number and strength. Most studies of long-term synaptic plasticity have focused on synapse-specific forms of plasticity such as long-term potentiation (LTP). While LTP is likely important for the refinement of neuronal circuitry, it probably not sufficient, because it tends to destabilize the activity of neuronal networks. We have concentrated our efforts on understanding the synaptic plasticity mechanisms that stabilize neuronal activity in the face of ongoing changes in synapse number and strength. For the past several years we have been studying the mechanism and function of synaptic scaling - a form of homeostatic synaptic plasticity - using a culture system derived from postnatal visual cortex. Despite increasing study of homeostatic synaptic plasticity over the past 4 years, there are many unanswered questions about its mechanism and function. In this renewal application we wish to examine the involvement of presynaptic and postsynaptic mechanisms in the expression of synaptic scaling, and to disentangle the relative contributions of presynaptic and postsynaptic activity in the induction of synaptic scaling. We will use a combination of electrophysiological, quantitative immunohistochemical, and time-lapse imaging methods to quantify changes in postsynaptic AMPA receptor accumulation. Using RNAi to selectively reduce expression of individual AMPAR subunits we will determine which subunits are targeted for insertion during synaptic scaling. We will determine whether there are complementary changes in presynaptic transmitter release, and whether postsynatpic and presynaptic mechanisms are temporally and causally related or occur independently. Finally, we will ask whether synaptic scaling is induced by local changes in efficacy of presynaptic inputs, or is a global function of postsynaptic activity. In addition to generating important mechanistic insights into activity-dependent plasticity, these experiments will test some fundamental assumptions about the function of synaptic scaling.